Development of an LC⁻Tandem Mass Spectrometry Method for the Quantitative Analysis of Hercynine in Human Whole Blood.

Given that the peculiar redox behavior of ergothioneine involves a rapid regeneration process, the measurement of its precursor and redox metabolite hercynine could be particularly useful in assessing its role in oxidative stress or other biological processes. Thus, a LC-MS/MS method for the determination of hercynine concentrations in whole blood was developed. After lysis of red blood cells by cold water, samples were filtered on micro concentrators at a controlled temperature of 4 °C. The clear filtered fluid was then treated with diethylpyrocarbonate to derivatize hercynine for the analysis by LC-MS/MS. The derivatized analyte was isocratically separated as a carbethoxy derivative on a C18 column with a mobile phase of an aqueous 0.1% v/v formic acid and acetonitrile (95:5). Effluents were monitored by MRM transitions at m/z 270.28→95 and 273.21→95 for hercynine and its deuterated counterpart, respectively. No cross-talk between MRM transitions was observed and a good linearity was found within a range of 35⁻1120 nmol/L. The LOD and LOQ were, respectively, 10.30 and 31.21 nmol/L with an intraday and intermediate precision below 7%. The average hercynine concentration in whole blood from 30 healthy male volunteers (aged 77 ± 12 years) was 178.5 ± 118.1 nmol/L. Overall, the method is easy to perform, allowing a rapid and accurate assessment of whole blood concentrations of hercynine.

Identification of the Main Intermediate Precursor of l-Ergothioneine Biosynthesis in Human Biological Specimens.

A capillary electrophoresis coupled to tandem mass spectrometry (CE-MS/MS) has been used to make a qualitative determination of hercynine-the main precursor of l-ergothioneine biosynthesis-in some key human biological specimens, such as urine, whole blood, plasma, and saliva. From semiquantitative analysis results, the highest concentrations of hercynine were detected in saliva and whole blood, whereas much lower concentrations were measured in urine and plasma. Whole blood was the biological matrix with the highest concentration of l-ergothioneine followed by plasma, saliva, and urine. The antioxidant effects attributed to l-ergothioneine, along with its peculiar antioxidant mechanism, offer a possible explanation for the presence of the hercynine, as well as its concentration, in the considered biological matrices.

Hercynine, Ergothioneine and Redox State in Stallion's Seminal Plasma.

The dependence of a stallion's spermatozoa on oxidative phosphorylation for energy requirements results in an unconventional relationship between reactive oxygen species (ROS) production and fertility. In such a scenario, antioxidant activity must be finely controlled and not affect the essential functions of ROS. Some in vivo evidence suggests that the naturally occurring antioxidant ergothioneine (ERT) interferes with the critical roles of ROS/reactive nitrogen species (RNS) in pro-oxidant states but not in healthy tissues. The measurement of ERT in seminal plasma collected from 14 stallions (five Anglo-Arab, five Sella Italiano and four Thoroughbreds of which three are Arabian and one English) aged 16 ± 6 years (range 6-25 years) confirms that ERT is present at high concentrations in this biological fluid, between 16.80 and 971.48 µmol/L. Although the presence of high ERT concentrations in the seminal plasma of a stallion has long been known, its exact biological role is uncertain. This might be due to the peculiar antioxidant cycle of ERT, specifically its rapid recovery, which potentially masks concentration fluctuations and, therefore, the extent of its physiological effects. The measurement of the ERT precursor and redox metabolite hercynine (ERY) may overcome such issues, as ERY does not undergo regeneration processes. ERY was detectable and measurable in the seminal plasma of all stallions at a median concentration of 7.50 (IQR 15.26) nmol/L. The analysis of the association between the ERT and ERY, as well as with other established antioxidants such as glutathione and cysteine, suggests that ERT may play a major role in the antioxidant machinery of seminal plasma, and that ERY might serve as a new combined marker of oxidative stress and semen quality.